This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Time-resolved crystallography is most suited to follow reactions in real time and on the atomic length scale. It combines crystallography with chemical kinetics. Substantial effort has been made to collect data sets contiguous over several orders of magnitudes in time. From this the structures of the intermediates as well as the chemical kinetic mechanism may be deduced using advanced mathematical methods such as the singular value decomposition. However, it turns out, that several candidates of these mechanisms explain the data equally well, hence they are degenerate. To remove this degeneracy, an additional parameter has to be varied to determine a unique set of rate coefficients describing a unique mechanism. Since rate coefficients usually follow Arrhenius's law, the temperature needs to be varied. Hence time-resolved crystallography, which is 4-dimensional - 3 space dimensions plus the time- would become 5-dimensional. We will perform these kind of experiments for the first time using crystals of photo-active yellow protein, which was previously well characterized by means of spectroscopy and time-resolved crystallography. Using singular value decomposition, relaxation times for the structural transitions will be determined at several temperatures starting at -5 deg C up to 35 deg C using the time-series of crystallographic Laue data at each temperature. Ultimately, this will give a unique set of clearly distinguishable intermediates plus the corresponding mechanism of the PYP photocycle.